The objective of this project is to develop an additive solution optimized for red blood cells (RBCs) stored under hypoxic environment, with the goal of producing highest quality RBCs by minimizing storage- associated damage (storage lesion). The additive solution developed in this project will be commercialized as a part of the Hemanext hypoxic storage platform. The proposal builds on the strong preliminary data showing that hypoxic storage of RBC (i) decreases oxidant stress and improves energy metabolism through mechanisms of intracellular alkalinization and concomitant removal of oxygen, a rate-limiting substrate for pro-oxidant reactions; (ii) promotes energy metabolism by preventing the oxidation of functional residues in key enzymes such as glyceraldehyde 3-phosphate dehydrogenase; (iii) significantly outperforms control RBCs with respect to the two FDA gold standard to determine storage quality, hemolysis and post-transfusion recovery; (iv) improves resuscitation (superior microvascular perfusion and oxygen delivery) in hemorrhaged rats, at significant lower doses than control RBCs and performing comparably to fresh RBCs even when end of storage hypoxic RBCs are transfused. In addition, hypoxic RBC reduces variability of quality present in the current products, while also reducing agents implicated in adverse events in the recipients. The potential for the development of storage additives tailored towards the specific metabolic needs of RBCs stored in the absence of oxygen became apparent from the wealth of data generated to test hypoxic storage for commercialization. Such an additive is expected to further improve RBC storage quality and promote transfusion outcomes in animal models. In order to develop such an additive and test the superiority of hypoxic RBCs stored in this new additive, the proposed project combines Hemanext?s extensive work over 10 years of research and development of hypoxic RBC storage platform (in part funded by SBIR Phase I, II and IIB grants), and Omix Technologies? extensive published works on RBC metabolism in vivo and during blood bank storage. The three aims of this Phase II proposal are: First, to exploit innovative high-throughput metabolomics tools to optimize the formulation of five hypoxic additive solutions from candidates of 50+ additive formulations previously tested under normoxic storage. Additional formulations will be also examined that have been designed according to insights from RBC metabolism under hypoxic condition gathered from collaborative work over the past four years. Second, to scale up the development of the top performing additive formulation developed in aim 1, in order to determine storage quality under control and hypoxic storage in the new additive through the evaluation of hemolysis and 24-hour post-transfusion recovery studies in end of storage RBCs. Third, to further evaluate the potential efficacy of the top candidate additive solutions in a rodent model of hypovolemic shock and resuscitation. !